398
Anal. Chem. 1988, 58, 398-401
Combined Ion Selective Electrode and Fluorescence Quenching Detection for Copper-Dissolved Organic Matter Titrations Stephen E. Cabaniss and Mark S. Shuman* Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27514
Copper-dlssolved organlc matter blndlng Is observed for Black Lake, NC, fulvic acld by measuring free metal with a copper Ion selective electrode (ISE) and the unbound organk llgand by fluorescence. The two detectors agree at low, aquatic levels of copper loading; a dlscrepancy at high copper concentrations may result from preclpitatlon or lnapproprlate assumptions about the fluorescence technlque. Compiementary error propertles of combined detectlon provide iowerror titration data over a wider range of pH and pCu than either detector alone. Calibration by ISE Is more reliable than curve fitting for relating fluorescence to bound copper concentrations.
Most investigations of trace metal-dissolved organic matter (DOM) complexation employ titrimetry in which free metal M, free ligand L, or the metal-DOM complex ML concentration is monitored. Usually, free metal is measured and bound metal is calculated by subtracting free from total metal (I). Metal detection techniques include anodic stripping voltametry (ASV),ion selective electrode (ISE) potentiometry, and separation followed by ASV, atomic absorption spectrometry, or radiotracer methods. The cupric ISE, which responds to aquo Cu(II), has been used with freshwater DOM titrations (2-5). Simultaneous formation of inorganic copper complexes such as CuC03 and CuOH in freshwaters complicates ISE data analysis a t alkaline pH, since the concentrations of inorganic complexes, [MI], must be calculated and subtracted to obtain [ML] (5). Fluorescence quenching (FQ) is the only quantitatively tested ligand detection method (6, 7). It is rapid and sensitive, allowing measurements at natural DOM concentrations. Binding of paramagnetic ions like Cu2+near fluorophores quenches DOM fluorescence and the difference in fluorescence before and after metal addition is an estimate of bound ligand concentration, i.e. quenching = Q = Imag -I (1) bound ligand = Lt - [L]
(2)
bound ligand = AQ
(24
where I,, is the fluorescence intensity when Mt = 0, I is the fluorescence intensity after metal addition, and A is an empirical constant rehting quenching and complex concentration. The FQ technique is valid only if binding by quenchable ligands is proportional to binding by all ligands for all degrees of loading, so that A is indeed constant. Weber and co-workers found this requirement fulfilled for soil and water fulvic acid (6, 7). Since FQ does not require an estimate of [MI] to calculate [ML], it is easy to use in complex inorganic media such as seawater. In addition to analytical considerations, metal-DOM complexation studies require careful choice of a binding model 0003-2700/86/0358-0398$01.50/0
to analyze experimental data. DOM and its humic and fulvic fractions are complex mixtures of ligands. The number of different metal-binding sites and total site concentration cannot be determined a priori, and binding stoichiometryand site interactions are not well understood. Various equilibrium binding models proposed to aid experimental design and data interpretation are all compromises between rigor and practicality (8). Most models assume 1:l stoichiometry bound metal = [Mt] - [MI - [MI] bound ligand = Lt - [L] bound metal = bound ligand = [ML]
(4)
(where [Mt] is the total metal concentration, [MI is the free (aquo) metal concentration, [MI] is the sum of concentrations of all inorganic metal complexes, Lt is the sum of all individual binding site concentrations, [L] is the sum of all individual free binding site concentrations, and [ML] is the sum of all individual metal-DOM complex concentrations),which greatly simplifies data analysis; however, titration curves can also be fitted to mixed stoichiometry models (2, 8). Microscopic reaction quotients may be defined by postulating a number of discrete 1:l binding sites with different binding values
(5) for each ith binding site, bound metal [ML] may be calculated by
where [ML] = CiSln[MLi],Lt = Ci,lnLti, and n is the number of discrete sites. Titration data may be curve-fit by assuming n is 2 or 3, but goodness of fit criteria cannot be used to establish that only two or three discrete binding sites are present (3,8).Alternatively, the distribution of microscopic binding sites may be expressed as an affinity spectrum (9) or as a Gaussian distribution (3)and used to evaluate binding. A macroscopic mass action quotient, K,, may be calculated directly if [MI, [ML], and [L] are known.
(7)
K,,, is the sum of microscopic Kiweighted by [MLi]; it is a function of loading, pH, and other variables and is useful for evaluating binding trends Yith varying experimental conditions. A similar function, K , which includes a proton term in the numerator, has been used by Gamble and co-workers (10).
The work described here attempts to draw attention to the dual problems of experimental design and choice of models inherent in any metal-DOM binding study. The objectives 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986 25
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are (1)to demonstrate the advantages of simultaneous monitoring by ISE and FQ to improve error propagation in metal-DOM titrimetry and (2) to suggest practices that may improve reliability and expand applicability of FQ.
EXPERIMENTAL SECTION The DOM sample was a Black Lake fulvic acid, isolated by standard procedures of the International Humic Substances Society (11)and described elsewhere (11,12). Seawater titrations were performed on 0.45 wm filtered coastal seawater from near Charleston, SC. The samples were kept in the dark and under refrigeration up to 3 weeks until analysis. Copper solutions were made from Aldrich copper perchlorate hexahydrate and standardized vs. EDTA. The background electrolyte was sodium perchlorate, and pH was adjusted by additions of NaOH or HCIO1. Base titrant was made from solid sodium hydroxide and standardized vs. potassium acid phthalate. All chemicals were reagent grade. An Orion cupric ISE Model 94-29 and an Orion double-junction electrode Model 90-02 were used with an Orion Model 801millivolt pH meter to estimate cupric ion activity. The electrodes were stored in a Cu-EDTA buffer overnight and when not in use. Before each titration, the Cu ISE was cleaned in a 10% sulfuric acid solution and then thoroughlyrinsed (13). The ISE responded to light and O2 (4, 14), so titrations were run under constant illumination and 02-freeconditions. The millivolt signal was monitored by an Apple I1 microcomputerand readings were taken when the drift of the electrode was less than 0.1 mV/min (15); this required 2-30 min, depending on total copper concentrations. The electrode was polished weekly with silica strips supplied by Orion. A Baird Nova spectrofluorometer was used to monitor fluorescence of the Black Lake fulvic acid at excitation 350 nm, emission 450 nm, and a 10-nm excitation slit width and a 20-nm emission slit width. Eight-secondintegration time stabilized the signal to h0.3 units on a scale of 0.0 to 100.0. The fluorescence cell was thermostated at 25 "C. A Farrand Mark I spectrofluorometer was used to monitor seawater DOM fluorescence under identical conditions, except that signal stability was *0.2 units. Base Titrations. A solution for 12 mg of C/L of DOM and 0.1 M NaC104was adjusted to pH 4 with acid and purged 15 min with N2 Fluorescence,pH, and millivolts were measured for each addition of base. Quenching as a function of pH was calculated by fitting fluorescence intensity vs. pH as a piecewise polynomial and subtracting measured intensity (copper added) from interpolated values (no copper added). Copper Titrations. A solution containing 12 mg of C/L of DOM and 0.1 M NaClO, was purged with N2 or N2/C02 and adjusted to pH 5.00 or 6.00with lo4 M sodium acetate or sodium bicarbonate buffers, respectively. Copper additions were chosen to provide a 3 mV change in the Cu ISE signal for each of 40-60 data points. Occasional small additions of acid or base were necessary to maintain constant pH. After the millivolt reading stabilized, the pH, millivolt, and fluorescence intensity were recorded. Seawater DOM titrations were monitored only by fluorescence;a 5-min equilibration period was allowed after each copper addition.
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RESULTS Metal-into-DOM titrations of Black Lake DOM a t pH 5.0 and 6.0 reveal smooth, featureless curves by ISE or fluorescence detection (Figure 1). These curves are typical of such titrations, and may be fitted by a variety of binding models (2,8,10,16). Affinity spectra indicate a distribution of binding sites tending to higher concentrations for the weaker sites (Figure 2). Titrations of seawater DOM show smooth curves similar to those for Black Lake, but the seawater curves asymptotically approach a fluorescence intensity minimum a t much lower total copper concentrations. The calculated quantities, Mbnd = Mt - M , from the ISE and quenching, Q, from the fluorescence measurements have large errors when similar numbers are subtracted (Mt N M , I,,, I), with Mbnd error greatest at high loading and the Q error greatest a t the beginning of the titration. The error properties of the two techniques are clearly complementary (Figure 3). Q is plotted vs. M b n d in Figure 4 and is linear for M b n d
